WO2017176541A1 - Synthèse enzymatique d'acides nucléiques - Google Patents

Synthèse enzymatique d'acides nucléiques Download PDF

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Publication number
WO2017176541A1
WO2017176541A1 PCT/US2017/024939 US2017024939W WO2017176541A1 WO 2017176541 A1 WO2017176541 A1 WO 2017176541A1 US 2017024939 W US2017024939 W US 2017024939W WO 2017176541 A1 WO2017176541 A1 WO 2017176541A1
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Prior art keywords
nucleotide
reaction
inactive
dna polymerase
error prone
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PCT/US2017/024939
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English (en)
Inventor
Henry H. Lee
George M. Church
Reza Kalhor
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President And Fellows Of Harvard College
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Application filed by President And Fellows Of Harvard College filed Critical President And Fellows Of Harvard College
Priority to US16/090,640 priority Critical patent/US10870872B2/en
Priority to CN201780034503.2A priority patent/CN109312493B/zh
Publication of WO2017176541A1 publication Critical patent/WO2017176541A1/fr
Priority to US16/951,254 priority patent/US20210108243A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • the present invention relates in general to methods of making oligonucleotides and polynucleotides using enzymatic synthesis.
  • DNA has been proposed as a highly desirable medium for storage of digital information.
  • the barrier to such use of DNA is the low efficiency and speed as well as the high cost of current synthesis methods.
  • DNA is synthesized using phosphoramidite precursors in organic solvents. These chemical synthesis methods result in errors of approximately 1% and take approximately 10 minutes per addition step.
  • TdT terminal deoxynucleotidyl transferase
  • the present disclosure addresses this need and is based on the discovery of methods that synthesize nucleic acids of a desired sequence using a template-independent DNA polymerase.
  • the methods according to the disclosure sequentially expose nucleic acid polymers to nucleotide polymerization units (NPUs) that extend the polymer by an expected length.
  • NPUs nucleotide polymerization units
  • each NPU comprises terminal deoxynucleotidyl transferase (TdT) and one type of nucleotide substrate (such as A, C, G, and T/U).
  • TdT terminal deoxynucleotidyl transferase
  • A, C, G, and T/U nucleotide substrate
  • the nucleotide substrates are in the form of nucleotide triphosphates which are the active form for polymerization purposes as contemplated by the present disclosure.
  • the disclosure provides novel physical, chemical, and enzymatic methods to control NPU extensions and limit them to a few nucleotides. These novel methods overcome problems encountered under commonly used laboratory conditions in which NPUs would extend nucleic acid polymers indefinitely and uncontrollably. These novel methods provide sequential exposure of the DNA polymers to NPUs that contain different nucleotides and obtain nucleic acid polymers of a desired sequence, thus serving a basis for enzymatic encoding of digital information into DNA. These novel methods provide improved control of the number and nature of nucleotides that template-independent DNA polymerases, such as TdT, incorporate into nucleic acid polymers and enable user-defined synthesis of nucleic acid sequences useful for biological applications.
  • template-independent DNA polymerases such as TdT
  • TdT terminal deoxynucleotidyl transferase
  • a unique DNA polymerase which extends single-stranded oligonucleotides.
  • TdT is template-independent, a property that enables the incorporation of bases into a growing strand of nucleic acids based on availability of provided nucleotides. This property makes TdT an attractive DNA polymerase for de novo DNA synthesis.
  • the disclosure provides that under ideal circumstances, it is desirable to limit the number of additions by TdT to one.
  • DNA is not only suitable for digital information storage but also for use in biological/genetic application.
  • the disclosure further provides that limiting the additions to one is not necessarily required for storage of information into DNA.
  • An exemplary proper encoding strategy that, instead of considering each base as a unit of information, considers each stretch of one or more identical bases (i.e., a homopolymer) as a unit of information can be used for digital storage purposes. For instance, if every stretch of A or T represents 0 and every stretch of C or G represents 1 , the sequence
  • the present disclosure provides a method for making a polynucleotide comprising (a) delivering a reaction reagent mobile phase including at least an error prone template independent DNA polymerase, a selected nucleotide triphosphate and cations along a fluidic channel to a reaction site, wherein the reaction site includes an initiator attached thereto and having a 3' terminal nucleotide, wherein reaction reagents are present in the reaction reagent mobile phase at selected concentrations, wherein the reaction reagent mobile phase has a selected volume and a selected flow rate to achieve a selected residence time at the reaction site under conditions which covalently add one or more of the selected nucleotide to the 3' terminal nucleotide such that the selected nucleotide becomes a 3' terminal nucleotide, (b) delivering an organic wash mobile phase to the reaction site at a fluid flow rate to remove the reaction reagents from the reaction site, and (c) repeating steps (a) and (b) until the polynucle
  • the present disclosure provides a method for making a polynucleotide comprising (a) combining a selected nucleotide triphosphate, cations, an error prone or template independent DNA polymerase, and a nucleotide triphosphate inactivating enzyme at a reaction site including an initiator sequence attached thereto and having a 3' terminal nucleotide, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of the selected nucleotide to the 3' terminal nucleotide such that the selected nucleotide becomes a 3' terminal nucleotide and under conditions which inactivate free nucleotide triphosphates until free nucleotide triphosphates are substantially inactivated, wherein a desired number of the selected nucleotide is added to the initiator sequence, and (b) repeating step (a) until the polynucleotide is formed.
  • the present disclosure provides a method for making a polynucleotide comprising (a) combining a selected inactive nucleotide, cations, an error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3' terminal nucleotide, activating the selected inactive nucleotide, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of a selected activated nucleotide to the 3' terminal nucleotide such that the selected activated nucleotide becomes a 3' terminal nucleotide and under conditions wherein a desired number of the selected activated nucleotide is added to the initiator sequence, and (b) repeating step (a) until the polynucleotide is formed.
  • the present disclosure also provides a method for making a polynucleotide comprising (a) combining a selected nucleotide triphosphate, cations, an inactive error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3' terminal nucleotide, activating the inactive error prone or template independent DNA polymerase, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of a selected nucleotide to the 3' terminal nucleotide such that the selected nucleotide becomes a 3' terminal nucleotide and under conditions wherein a desired number of the selected nucleotide is added to the initiator sequence, and (b) repeating step (a) until the polynucleotide is formed.
  • the present disclosure provides a method for making a polynucleotide comprising (a) combining a selected nucleotide triphosphate, cations, an error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3' terminal nucleotide, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of a selected nucleotide to the 3' terminal nucleotide such that the selected nucleotide becomes a 3' terminal nucleotide and wherein the error prone or template independent DNA polymerase is inactivated to terminate addition of the selected nucleotide, and (b) repeating step (a) until the polynucleotide is formed.
  • the present disclosure further provides a method for making a polynucleotide comprising (a) combining a selected inactive nucleotide, cations, an inactive error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3 ' terminal nucleotide, activating the nucleotide and activating the error prone or template independent DNA polymerase, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of a selected nucleotide to the 3' terminal nucleotide such that the selected nucleotide becomes a 3' terminal nucleotide, and (b) repeating step (a) until the polynucleotide is formed.
  • FIGS. 1A & IB depict in schematic a physical control method of NPU exposure to nucleic acid polymers in a fluidic device.
  • FIG. 1A depicts in schematic a top-down view of sequence of NPUs as they flow over the initiator oligo patch.
  • FIG. IB depicts in schematic a microfluidic device which implements this physical control.
  • FIGS. 2 A & 2B depict in schematic a chemical control method of NPU exposure to nucleic acid polymers in a fluidic device.
  • FIG. 2A depicts in schematic NPU in a solution with TdT and apyrase covering an initiator oligo.
  • FIG. 2B depicts in schematic polymerization that occurs when each base is deposited onto the NPU.
  • FIG. 3 depicts screened nucleotide analog substrates for TdT to select for better performance on extension efficiency, rate, and extension length distribution according to the embodiments of the disclosed methods.
  • FIG. 4 depicts in schematic an apparatus which implements certain embodiments of the disclosed methods for TdT-based NPU synthesis of DNA with given information content.
  • Initiator DNA is immobilized to a glass surface and the desired nucleotide is deposited on the DNA.
  • NPUs including TdT and apyrase are deposited to catalyze a limited extension. After cleanup, the oligo is ready for the deposition of the next nucleotide and NPUs.
  • FIG. 5 depicts in schematic a PDMS mask template for generating a microfluidic device which implements the physical control of NPU exposure to nucleic acid polymers.
  • FIG. 6 depicts PCR products on Agarose gel according to the embodiments of the disclosed methods.
  • FIG. 7 depicts quantification of the number of nucleotides added to the oligonucleotide initiator for each nucleotide type and concentration in addition to the number of oligonucleotide initiators that received nonzero addition of nucleotides for each nucleotide type and concentration according to the embodiments of the disclosed methods and as measured by high-throughput single-molecule sequencing.
  • FIG. 8 depicts fraction of initiators with nonzero addition of nucleotides per nucleotide concentration according to the embodiments of the disclosed methods.
  • FIG. 9 depicts 3 '-modified reversible terminators.
  • FIG. 10 depicts 3'-modified reversible terminators.
  • FIG. 11 depicts base-modified nucleotides.
  • FIG. 12 depicts a schematic of the divalent cations that change polymerization mechanism and thereby efficiency and kinetics of polymerization.
  • FIG. 13 depicts results for pH regulation of TdT enzyme activity on a TBE-Urea gel according to the embodiments of the disclosed methods.
  • FIG. 14 depicts results for reversible pH regulation of TdT enzyme activity on a TBE- Urea gel according to the embodiments of the disclosed methods.
  • FIG. 15 is a gel image showing results for 5BR-dCTP and 5I-dCTP.
  • FIG. 16 is a gel image showing results for 5h-dCTP and 5hm-dCTP.
  • FIG. 17 is a gel image showing results for 5m-dCTP and dCTP.
  • the present disclosure provides methods of modulating activity of components and reagents used in template independent nucleic acid synthesis, such as nucleotides, template independent polymerase, and cations.
  • the residence time of each of these components at a reaction site can be altered to modulate addition of a nucleotide to an initiator sequence or a growing nucleic acid chain.
  • Each of these components can be activated or deactivated to modulate the addition of a nucleotide to an initiator sequence or growing nucleic acid chain.
  • nucleotide addition can be controlled to a desired number of nucleotides, such as one nucleotide, two nucleotides, three nucleotides etc.
  • the disclosure provides that addition is limited to one nucleotide, two nucleotides, three nucleotides or more during one round of nucleotide addition.
  • This activation or inactivation of the reaction components may be reversible to allow for multiple rounds of nucleotide polymerization that each adds a different nucleotide to the primer or growing polynucleotide chain.
  • the present disclosure provides methods of "mobile-phase oligonucleotide synthesis,” an enzymatic synthesis that enables control over the number and nature of nucleotides that an error prone or template independent polymerase such as TdT adds to a primer strand of DNA, i.e., the primer/initiator for DNA synthesis.
  • the methods involve a fluidic/microfluidic device wherein initiator oligonucleotides (nucleic acids that act as the initial substrate for TdT to extend with the desired sequence) are immobilized on the surface of this device in a patch (i.e., the initiator patch).
  • the patch is then exposed to "packets" of reagents that include TdT pre-mixed with one of the four possible nucleotide triphosphates (dNTPs).
  • the microfluidic device accurately controls the exact exposure time of the patch to each TdT- dNTP packet, thus limiting the addition to a desired count or a desired distribution of counts.
  • the device also allows control over the order of packets, thereby enabling control over the incorporated sequence. For instance, for the synthesis of the sequence "GATC,” the patch will be exposed to a packet of TdT-dGTP, followed by a packet of TdT-dATP, followed by a packet of TdT-TTP, followed by a packet of TdT-dCTP wherein the patch is exposed to each packet for an optimal time that ensures single additions or additions of a desired length or length distribution by the enzyme.
  • the disclosure provides a variety of ways to achieve precise control over the number of nucleotides that are added by TdT.
  • the disclosure provides "kinetic" control wherein each packet resides over the patch for a period of time long enough for a single addition but too short for two or more additions.
  • the disclosure provides combining the kinetic control with various chemical and biochemical approaches to achieve control over the number of additions of nucleotides that are added by TdT.
  • reversible terminator dNTPs can be used instead of natural dNTPs. Terminator dNTPs are modified dNTPs that TdT can add to a growing DNA primer but cannot extend further.
  • a packet that reverts the termination chemically, physically, or enzymatically will also be introduced, followed by the next desired reversible terminator dNTP-TdT packet, and so on.
  • controlling the number of additions is achieved by using an engineered TdT or packet composition wherein the TdT enzyme remains bound to the primer at some stage in its catalytic cycle, thus blocking further additions.
  • the next packet can allow the enzyme to complete the catalytic cycle and detach but lack any dNTPs so unwanted additions can be prevented.
  • an important property of the mobile-phase synthesis strategy is that more than one liquid phase can be used in the microfluidic device.
  • an inert organic phase such as mineral oil
  • An organic phase between the packets also ensures a sharp packet border and thus achieves very precise control over the exposure time of the patch to each packet.
  • active chemicals in this organic phase that for instance reverse termination by terminator nucleotides is also contemplated.
  • the present disclosure provides that another important property of the mobile-phase synthesis strategy is that it allows a different condition to be used in each of the four TdT- dNTP packet types. This is important as the kinetics of the enzyme may be different for different dNTPs. Thus, to obtain optimal results, different conditions, such as type and concentration of divalent ions may need to be used for different dNTPs.
  • the present disclosure provides methods of the mobile -phase oligonucleotide synthesis which enable rapid and high-accuracy synthesis of custom DNA sequences by the template-independent DNA-polymerase terminal deoxynucleotidyl transferase (TdT).
  • the methods according to the present disclosure can be used for synthesis of cheaper, more accurate and longer custom DNA sequences for various biochemical, biomedical, or biosynthetic applications. Furthermore, given the potential for high-speed DNA synthesis, the methods according to the present disclosure can facilitate the use of DNA as an information storage medium. In this case, a solid-phase synthesis device can be used to record digital information in DNA molecules.
  • Embodiments of the disclosure are directed to a method for making a polynucleotide wherein addition of the nucleotides can be physically controlled.
  • the method comprises (a) delivering a reaction reagent mobile phase including at least an error prone template independent DNA polymerase, a selected nucleotide triphosphate and cations along a fluidic channel to a reaction site, wherein the reaction site includes an initiator attached thereto and having a 3' terminal nucleotide, wherein reaction reagents are present in the reaction reagent mobile phase at selected concentrations, wherein the reaction reagent mobile phase has a selected volume and a selected flow rate to achieve a selected residence time at the reaction site under conditions which covalently add one or more of the selected nucleotide to the 3' terminal nucleotide such that the selected nucleotide becomes a 3' terminal nucleotide, (b) delivering an organic wash mobile phase to the reaction site at a fluid flow rate to remove the reaction reagents
  • the selected volume and selected flow rate for the reaction reagent mobile phase is determined based on reactivity of the selected nucleotide triphosphate present in the reaction reagent mobile phase. In another embodiment, the selected volume and selected flow rate for the reaction reagent mobile phase differ based on the selected nucleotide triphosphate present in the reaction reagent mobile phase. In certain embodiment, the selected flow rate for the reaction reagent mobile phase is constant and the selected volume differs based on the selected nucleotide triphosphate present in the reaction reagent mobile phase. In one embodiment, the selected volume of the reaction reagent mobile phase is constant and the selected flow rate differs based on the selected nucleotide triphosphate present in the reaction reagent mobile phase.
  • the selected flow rate for the reaction reagent mobile phase is constant and the selected volume differs based on the selected nucleotide triphosphate present in the reaction reagent mobile phase and the desired number of the selected nucleotides to be added to the 3 ' end of the polynucleotide.
  • the selected volume of the reaction reagent mobile phase is constant and the selected flow rate differs based on the selected nucleotide triphosphate present in the reaction reagent mobile phase and the desired number of the selected nucleotides to be added to the 3' end of the polynucleotide.
  • the reaction site is a surface area on the surface of the fluidic channel.
  • the selected concentration of reaction reagents in the reaction reagent mobile phase is determined by the selected nucleotide triphosphate present in the reaction reagent mobile phase.
  • the reaction site is within the fluidic channel.
  • the reaction site is a structure within the fluidic channel.
  • the reaction site is a collection of beads within the fluidic channel.
  • the reaction site is an electrode on the surface of the fluidic channel.
  • the reaction site is an electrode within the fluidic channel.
  • the initiator includes one or more nucleotides.
  • the residence time is sufficient to limit the number of covalent additions of the selected nucleotide.
  • the organic wash mobile phase is immiscible with the reaction reagent mobile phase.
  • the reaction reagent mobile phase is bounded on either end by an organic wash mobile phase.
  • the organic wash mobile phase inactivates the reaction reagent mobile phase at the reaction site.
  • an air plug is used instead of or in addition to the organic wash mobile phase.
  • an aqueous wash mobile phase is used instead of or in addition to the organic wash mobile phase.
  • an aqueous wash mobile phase is used instead of or in addition to an air plug.
  • a plurality of reaction reagent mobile phases bounded on either end by an organic wash mobile phase flow to the reaction site.
  • the method according to the present disclosure further includes the step of monitoring covalent addition of the selected nucleotide.
  • the error prone template independent DNA polymerase is terminal deoxynucleotide transferase.
  • the cations are one or more of Zn +2 , Co "1"2 , Mg "1"2 or Mn +2 .
  • the selected nucleotide is a natural nucleotide or a nucleotide analog.
  • the selected nucleotide is a member selected from the group consisting of
  • the reaction reagent mobile phase includes a buffer comprising a monovalent salt, a divalent salt, a buffering agent, and a reducing agent at a suitable pH and temperature.
  • the reaction reagent mobile phase includes a buffer comprising 10 to 20 mM tris-acetate, 20 to 50 mM potassium acetate, 5 to 8 mM magnesium acetate, 0.5 to 1.0 mM DTT and with a pH of about 2 to 12 and at a temperature of about 10 and 80°C.
  • the reaction reagent mobile phase includes a buffer comprising 14 mM tris-acetate, 35 mM potassium acetate, 7 mM magnesium acetate, 0.7 mM DTT and with a pH of about 7.9 and at a temperature of about 25°C.
  • Certain embodiment of the disclosure is directed to an initiator that is attached by a cleavable moiety.
  • the method according to the disclosure further comprises releasing the polynucleotide from the reaction site after the desired sequence of nucleotides has been added to the 3' end of the polynucleotide.
  • the method according to the disclosure further comprises releasing the polynucleotide from the reaction site using an enzyme, a chemical, light, heat or other suitable method or reagent.
  • the method according to the disclosure further comprises releasing the polynucleotide from the reaction site, collecting the polynucleotide, amplifying the polynucleotide and sequencing the polynucleotide.
  • Embodiments of the disclosure are directed to a method for making a polynucleotide wherein the addition of nucleotides can be chemically controlled via inactivating active nucleotide triphosphates using an enzyme.
  • the method comprises (a) combining a selected nucleotide triphosphate, cations, an error prone or template independent DNA polymerase, and a nucleotide triphosphate inactivating enzyme at a reaction site including an initiator sequence attached thereto and having a 3' terminal nucleotide, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of the selected nucleotide to the 3' terminal nucleotide such that the selected nucleotide becomes a 3' terminal nucleotide and under conditions which inactivate free nucleotide triphosphates until free nucleotide triphosphates are substantially inactivated, wherein a desired number of the selected nucleotide is added to the initiator sequence
  • the nucleotide inactivating enzyme is a nucleotide triphosphate degrading enzyme. In one embodiment, the nucleotide triphosphate inactivating enzyme is a nucleotide triphosphate degrading enzyme that degrades nucleotide triphosphates at a rate slower than rate of addition of nucleotides by the error prone or template independent DNA polymerase. In certain embodiment, the nucleotide triphosphate inactivating enzyme is a nucleotide triphosphate degrading enzyme present at a concentration that degrades nucleotide triphosphates at a rate slower than rate of addition of nucleotides by the present concentration of the error prone or template independent DNA polymerase. In some embodiments, the nucleotide triphosphate inactivating enzyme comprises ATP diphosphohydrolase, dNTP pyrophosphatases, dNTPases, and phosphatases.
  • the concentration of nucleotide triphosphate inactivating enzyme is modulated to control addition of one or more nucleotides.
  • the nucleotide triphosphate inactivating enzyme renders free nucleotide triphosphates inactive.
  • the nucleotide inactivating enzyme renders free nucleotide triphosphates inactive by degradation.
  • the nucleotide inactivating enzyme renders free nucleotide triphosphates inactive by polymerizing them with each other.
  • the reaction conditions present a competing reaction between addition of free nucleotide triphosphates to the initiator sequence and degradation of free nucleotide triphosphates.
  • the selected nucleotide is added to the reaction site including the initiator sequence having the terminal nucleotide, the error prone or template independent DNA polymerase and the nucleotide inactivating enzyme.
  • the error prone or template independent DNA polymerase and the nucleotide inactivating enzyme are added to the reaction site including the initiator sequence having the terminal nucleotide, and the selected nucleotide.
  • the nucleotide inactivating enzyme is added to the reaction site including the initiator sequence having the terminal nucleotide, the error prone or template independent DNA polymerase and the selected nucleotide under conditions where the polymerase is inactive, and wherein the polymerase is activated upon addition of the nucleotide inactivating enzyme.
  • step (b) is repeated a plurality of times after which the reaction reagents are removed from the reaction site and additional reaction reagents are provided to the reaction site.
  • the reaction reagents are removed from the reaction site and additional reaction reagents are provided to the reaction site after each round of addition.
  • the reaction reagents are removed from the reaction site and additional reaction reagents are provided to the reaction site after each round of addition.
  • Embodiments of the disclosure are directed to a method for making a polynucleotide wherein the addition of nucleotides can be chemically controlled via activating inactive nucleotides.
  • the method comprises (a) combining a selected inactive nucleotide, cations, an error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3' terminal nucleotide, activating the selected inactive nucleotide, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of a selected activated nucleotide to the 3 ' terminal nucleotide such that the selected activated nucleotide becomes a 3' terminal nucleotide and under conditions wherein a desired number of the selected activated nucleotide is added to the initiator sequence, and (b) repeating step (a) until the polynucleotide is formed.
  • the inactive nucleotide is rendered active by a chemical reaction, an enzyme, heat, light or pH.
  • the inactive nucleotide includes a protecting group and the protecting group is removed.
  • the inactive nucleotide comprises NPE or DMNPE-caged nucleotides or similar caged nucleotides that are activated by light, heat, an enzyme, a chemical reaction, or pH.
  • the NPE-caged nucleotides comprise deoxynucleotide 5'-Triphosphate, P3-(l-(2- Nitrophenyl)Ethyl) esters.
  • the DMNPE-caged nucleotides comprise deoxynucleotide 5'-Triphosphate, 3-(l-(4,5-Dimethoxy-2-Nitrophenyl)ethyl) esters.
  • the selected inactive nucleotide is a nucleoside, nucleotide monophosphate, or nucleotide diphosphate form that is rendered into the active nucleotide triphosphate form by an activating enzyme such as nucleotide diphosphate kinase.
  • the inactive nucleotide is rendered active at a rate which allows addition of one or more activated nucleotides.
  • the inactive nucleotide is rendered active at a rate which allows addition of one or more activated nucleotides after which either the activated nucleotides or the polymerase is rendered inactive.
  • the inactive nucleotide is rendered active allowing addition of one or more activated nucleotides after which the polymerase is rendered inactive.
  • the polymerase is rendered inactive by a chemical reaction, divalent cations, an enzyme, heat, light or pH.
  • the selected inactive nucleotide is added to the reaction site including the initiator sequence having the terminal nucleotide, and the error prone or template independent DNA polymerase and the inactive nucleotide is activated.
  • step (b) is repeated a plurality of times after which the reaction reagents are removed from the reaction site and additional reaction reagents are provided to the reaction site.
  • Embodiments of the disclosure are directed to a method for making a polynucleotide wherein the addition of nucleotides can be chemically and enzymatically controlled via activating an inactive polymerase.
  • the method comprises (a) combining a selected nucleotide triphosphate, cations, an inactive error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3' terminal nucleotide, activating the inactive error prone or template independent DNA polymerase, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of a selected nucleotide to the 3' terminal nucleotide such that the selected nucleotide becomes a 3' terminal nucleotide and under conditions wherein a desired number of the selected nucleotide is added to the initiator sequence, and (b) repeating step (a) until the polynucleotide is formed.
  • the inactive error prone or template independent DNA polymerase is rendered active by a chemical reaction, divalent cations, an enzyme, heat, light or pH. In some embodiments, the inactive error prone or template independent DNA polymerase is rendered active by a chemical reaction, an enzyme, heat, light or pH and rendered inactive again by a chemical reaction, an enzyme, heat, light or pH after addition of the desired number of the selected nucleotide onto the initiator. In one embodiment, the inactive error prone or template independent DNA polymerase includes a protecting group and the protecting group is removed. In certain embodiment, the protecting group comprises a chemical group that is incorporated into the polymerase and is removable by light, heat, pH, or enzymes to control the polymerase activity.
  • the inactive error prone or template independent DNA polymerase is rendered active at a rate which allows addition of one or more nucleotides. In another embodiment, the inactive error prone or template independent DNA polymerase is rendered active at a rate which allows addition of one or more nucleotides after which either the nucleotides or the polymerase is rendered inactive. In one embodiment, the inactive error prone or template independent DNA polymerase is rendered active allowing addition of one or more nucleotides after which the polymerase is rendered inactive. In one embodiment, the polymerase is rendered inactive by a chemical reaction, an enzyme, heat, light or pH.
  • the inactive error prone or template independent DNA polymerase is added to the reaction site including the initiator sequence having the terminal nucleotide, and the selected nucleotide triphosphate and wherein the inactive error prone or template independent DNA polymerase is activated.
  • step (b) is repeated a plurality of times after which the reaction reagents are removed from the reaction site and additional reaction reagents are provided to the reaction site.
  • Embodiments of the disclosure are directed to a method for making a polynucleotide wherein the addition of nucleotides can be chemically and enzymatically controlled via inactivating active polymerase.
  • the method comprises (a) combining a selected nucleotide triphosphate, cations, an error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3' terminal nucleotide, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of a selected nucleotide to the 3' terminal nucleotide such that the selected nucleotide becomes a 3 ' terminal nucleotide and wherein the error prone or template independent DNA polymerase is inactivated to terminate addition of the selected nucleotide, and (b) repeating step (a) until the polynucleotide is formed.
  • the inactive error prone or template independent DNA polymerase is rendered active by a chemical reaction, an enzyme, heat, light or pH.
  • the active error prone or template independent DNA polymerase is rendered inactive by a chemical reaction, an enzyme, heat, light or pH after the addition of a desired number of the selected nucleotide and rendered active again by a chemical reaction, an enzyme, heat, light or pH for the addition of the next selected nucleotide to the 3' terminal nucleotide of the polynucleotide.
  • the inactive error prone or template independent DNA polymerase includes a protecting group and the protecting group is removed.
  • the protecting group comprises a chemical group that is incorporated into the polymerase and is removable by light, heat, pH, or enzymes to control the polymerase activity.
  • the error prone or template independent DNA polymerase is rendered inactive at a rate which allows addition of one or more nucleotides. In another embodiment, the error prone or template independent DNA polymerase is rendered inactive at a rate which allows addition of one or more nucleotides. In yet another embodiment, the error prone or template independent DNA polymerase is added to the reaction site including the initiator sequence having the terminal nucleotide, and the selected nucleotide triphosphate and wherein the error prone or template independent DNA polymerase is rendered inactive. In some embodiments, step (b) is repeated a plurality of times after which the reaction reagents are removed from the reaction site and additional reaction reagents are provided to the reaction site.
  • Embodiments of the disclosure are directed to a method for making a polynucleotide wherein the addition of nucleotides can be chemically and enzymatically controlled via activating the nucleotide and the polymerase.
  • the method comprises (a) combining a selected inactive nucleotide, cations, an inactive error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3' terminal nucleotide, activating the nucleotide and activating the error prone or template independent DNA polymerase, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of a selected nucleotide to the 3' terminal nucleotide such that the selected nucleotide becomes a 3' terminal nucleotide, and (b) repeating step (a) until the polynucleotide is formed.
  • either the active nucleotide or the active error prone or template independent DNA polymerase is rendered inactive to terminate addition of the selected nucleotide.
  • the active error prone or template independent DNA polymerase is rendered inactive by a chemical reaction, an enzyme, heat, light or pH after the addition of a desired number of the selected nucleotide and rendered active again by a chemical reaction, an enzyme, heat, light or pH for the addition of the next selected nucleotide to the 3' terminal nucleotide of the polynucleotide.
  • the inactive error prone or template independent DNA polymerase is rendered active by a chemical reaction, an enzyme, heat, light or pH.
  • the inactive error prone or template independent DNA polymerase includes a protecting group and the protecting group is removed.
  • the protecting group comprises a chemical group that is incorporated into the polymerase and is removable by light, heat, pH, or enzymes to control the polymerase activity.
  • the inactive nucleotide is rendered active by a chemical reaction, an enzyme, heat, light or pH.
  • the inactive nucleotide includes a protecting group and the protecting group is removed.
  • the inactive nucleotide comprises NPE-caged nucleotides or similar caged nucleotides that are removed by light, heat, an enzyme, a chemical reaction, or pH.
  • the NPE-caged nucleotides comprise deoxynucleotide 5'-Triphosphate, P3-(l-(2-Nitrophenyl)Ethyl) esters.
  • an activating enzyme such as nucleotide diphosphate kinase.
  • either the inactive nucleotide or inactive error prone or template independent DNA polymerase is rendered active at a rate which allows addition of one or more nucleotides.
  • step (b) is repeated a plurality of times after which the reaction reagents are removed from the reaction site and additional reaction reagents are provided to the reaction site.
  • Polymerases including without limitation error-prone or template-dependent polymerases, modified or otherwise, can be used to create nucleotide polymers having a random or known or desired sequence of nucleotides.
  • Template-independent polymerases can be used to create the nucleic acids de novo.
  • Ordinary nucleotides are used, such as A, T/U, C or G.
  • Nucleotides may be used which lack chain terminating moieties. Reversible terminators may be used in the methods of making the nucleotide polymers.
  • a template independent polymerase may be used to make the nucleic acid sequence. Such template independent polymerase may be error-prone which may lead to the addition of more than one nucleotide resulting in a homopolymer.
  • Oligonucleotide sequences or polynucleotide sequences are synthesized using an error prone polymerase, such as template independent error prone polymerase, and common or natural nucleic acids, which may be unmodified.
  • Initiator sequences or primers are attached to a substrate, such as a silicon dioxide substrate, at various locations whether known, such as in an addressable array, or random.
  • Reagents including at least a selected nucleotide, a template independent polymerase and other reagents required for enzymatic activity of the polymerase are applied at one or more locations of the substrate where the initiator sequences are located and under conditions where the polymerase adds one or more than one or a plurality of the nucleotide to the initiator sequence to extend the initiator sequence.
  • the nucleotides (“dNTPs") may be applied or flow in periodic applications. Nucleotides with blocking groups or reversible terminators can be used with the dNTPs under reaction conditions that are sufficient to limit or reduce the probability of enzymatic addition of the dNTP to one dNTP, i.e.
  • one dNTP is added using the selected reaction conditions taking into consideration the reaction kinetics. Nucleotides with blocking groups or reversible terminators are known to those of skill in the art. According to an additional embodiment when reaction conditions permit, more than one dNTP may be added to form a homopolymer run when common or natural nucleotides are used with a template independent error prone polymerase.
  • Polymerase activity may be modified using protease, photo-chemical or electrochemical modulation as a reaction condition so as to minimize addition of dNTP beyond a single dNTP.
  • a wash is then applied to the one or more locations to remove the reagents. The steps of applying the reagents and the wash are repeated until desired nucleic acids are created.
  • the reagents may be added to one or more than one or a plurality of locations on the substrate in series or in parallel or the reagents may contact the entire surface of the support, such as by flowing the reagents across the surface of the support.
  • the reaction conditions are determined, for example based on reaction kinetics or the activity of the polymerase, so as to limit the ability of the polymerase to attach more than one nucleotide to the end of the initiator sequence or the growing oligonucleotide.
  • polymerases can be modulated to be light sensitive for light based methods.
  • light is modulated to tune the polymerase to add only a single nucleotide.
  • the light is shone on individual locations or pixels of the substrate where the polymerase, the nucleotide and appropriate reagents and reaction conditions are present.
  • a nucleotide is added to an initiator sequence or an existing nucleotide as the polymerase is activated by the light.
  • polymerase activity can be controlled by pH. It is well known to a skilled in the art that each polymerase has an active pH range outside of which it is inactive.
  • the reaction reagent pH can adjusted in and out of the active range to control the polymerase.
  • TdT it has been determined that TdT is active below pH 10 but is inactive at pH 11. Therefore, if the initial setup of the reaction is at pH 11, temporarily changing the pH to anywhere below 10 can temporarily activate the TdT enzyme.
  • divalent cations such as Mg++, Co++, Mn++, Zn++, Ni++, are also known to a skilled in that art to be necessary for the activity of all known DNA polymerases. Chelating divalent cations from the reaction can stop the polymerase, or releasing divalent cations into the reaction can activate the polymerase.
  • Engineered polymerases can be created which are made active by a certain wavelength of light and made inactive by another wavelength of light. Such polymerases can contain light-reactive groups such as Azobenzene, Spiropyran, or Retinal. Engineered polymerases can be made that are rendered inactive at a certain temperature but are reactivated at another. Natural polymerases are also known to have this quality but to a limited and less useful level as compared to engineered polymerases.
  • Acyclovir Zaovirax
  • Zovirax a competitive, non-competitive, or uncompetitive chemical inhibitor of the polymerase
  • a flow cell or other channel such a microfluidic channel or microfluidic channels having an input and an output is used to deliver mobile phase or reaction fluids including reagents, such as a polymerase, a nucleotide and other appropriate reagents and washes to particular locations on a substrate within the flow cell, such as within a microfluidic channel.
  • reagents such as a polymerase, a nucleotide and other appropriate reagents and washes
  • reaction conditions will be based on dimensions of the substrate reaction region, reagents, concentrations, reaction temperature, and the structures used to create and deliver the reagents and washes.
  • pH and other reactants and reaction conditions can be optimized for the use of TdT to add a dNTP to an existing nucleotide or oligonucleotide in a template independent manner.
  • a dNTP to an existing nucleotide or oligonucleotide in a template independent manner.
  • reagents and reaction conditions for dNTP addition such as initiator size, divalent cation and pH.
  • TdT was reported to be active over a wide pH range with an optimal pH of 6.85. Methods of providing or delivering dNTP, rNTP or rNDP are useful in making nucleic acids.
  • nucleic acid molecule As used herein, the terms “nucleic acid molecule,” “nucleic acid sequence,” “nucleic acid fragment” and “oligomer” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides that may have various lengths, including either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • nucleic acid molecule In general, the terms “nucleic acid molecule,” “nucleic acid sequence,” “nucleic acid fragment,” “oligonucleotide” and “polynucleotide” are used interchangeably and are intended to include, but not limited to, a polymeric form of nucleotides that may have various lengths, either deoxyribonucleotides (DNA) or ribonucleotides (RNA), or analogs thereof.
  • DNA deoxyribonucleotides
  • RNA ribonucleotides
  • a oligonucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA).
  • deoxynucleotides such as dATP, dCTP, dGTP, dTTP
  • rNTPs ribonucleotide triphosphates
  • rNDPs ribonucleotide diphosphates
  • oligonucleotide sequence is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself.
  • This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • Oligonucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleotides.
  • the present disclosure contemplates any deoxyribonucleotide or ribonucleotide and chemical variants thereof, such as methylated, hydroxymethylated or glycosylated forms of the bases, and the like.
  • natural nucleotides are used in the methods of making the nucleic acids. Natural nucleotides lack chain terminating moieties.
  • modified nucleotides include, but are not limited to diaminopurine, S2T, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- acetylcytosine, 5 -(carboxyhydroxylmethyl)uracil, 5 -carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueos
  • Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and/or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone.
  • Nucleic acid molecules may also contain amine-modified groups, such as aminoallyl-dUTP (aa-dUTP) and aminohexhylacrylamide- dCTP (aha-dCTP) to allow covalent attachment of amine reactive moieties, such as N- hydroxy succinimide esters (NHS).
  • Alternatives to standard DNA base pairs or RNA base pairs in the oligonucleotides of the present disclosure can provide higher density in bits per cubic mm, higher safety (resistant to accidental or purposeful synthesis of natural toxins), easier discrimination in photo-programmed polymerases, or lower secondary structure.
  • Such alternative base pairs compatible with natural and mutant polymerases for de novo and/or amplification synthesis are described in Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A (2012) KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry, Nature Chem. Biol.
  • polymerases are used to build nucleic acid molecules, such as for representing information which is referred to herein as being recorded in the nucleic acid sequence or the nucleic acid is referred to herein as being storage media.
  • Polymerases are enzymes that produce a nucleic acid sequence, for example, using DNA or RNA as a template. Polymerases that produce RNA polymers are known as RNA polymerases, while polymerases that produce DNA polymers are known as DNA polymerases. Polymerases that incorporate errors are known in the art and are referred to herein as an "error-prone polymerases". Template independent polymerases may be error prone polymerases.
  • Error-prone polymerases will either accept a non-standard base, such as a reversible chain terminating base, or will incorporate a different nucleotide, such as a natural or unmodified nucleotide that is selectively given to it as it tries to copy a template.
  • Template-independent polymerases such as terminal deoxynucleotidyl transferase (TdT), also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase create nucleic acid strands by catalyzing the addition of nucleotides to the 3' terminus of a DNA molecule without a template.
  • TdT terminal deoxynucleotidyl transferase
  • DNTT DNA nucleotidylexotransferase
  • Cobalt is a cofactor, however the enzyme catalyzes reaction upon Mg and Mn administration in vitro.
  • Nucleic acid initiators may be 4 or 5 nucleotides or longer and may be single stranded or double stranded. Double stranded initiators may have a 3' overhang or they may be blunt ended or they may have a 3' recessed end.
  • TdT like all DNA polymerases, also requires divalent metal ions for catalysis.
  • TdT is unique in its ability to use a variety of divalent cations such as Co2+, Mn2+, Zn2+ and Mg2+.
  • the extension rate of the primer p(dA)n (where n is the chain length from 4 through 50) with dATP in the presence of divalent metal ions is ranked in the following order: Mg2+ > Zn2+ > Co2+ > Mn2+.
  • each metal ion has different effects on the kinetics of nucleotide incorporation.
  • Mg2+ facilitates the preferential utilization of dGTP and dATP whereas Co2+ increases the catalytic polymerization efficiency of the pyrimidines, dCTP and dTTP.
  • Zn2+ behaves as a unique positive effector for TdT since reaction rates with Mg2+ are stimulated by the addition of micromolar quantities of Zn2+. This enhancement may reflect the ability of Zn2+ to induce conformational changes in TdT that yields higher catalytic efficiencies. Polymerization rates are lower in the presence of Mn2+ compared to Mg2+, suggesting that Mn2+ does not support the reaction as efficiently as Mg2+. Further description of TdT is provided in Biochim Biophys Acta.
  • nucleotide pulse may replace Mg2+, Zn2+, Co2+, or Mn2+ in the nucleotide pulse with other cations designed modulate nucleotide attachment.
  • the nucleotide pulse replaces Mg++ with other cation(s), such as Na+, K+, Rb+, Be++, Ca++, or Sr++, then the nucleotide can bind but not incorporate, thereby regulating whether the nucleotide will incorporate or not.
  • a pulse of (optional) pre-wash without nucleotide or Mg++ can be provided or then Mg++ buffer without nucleotide can be provided.
  • the incorporation of specific nucleic acids into the polymer can be regulated.
  • these polymerases are capable of incorporating nucleotides independent of the template sequence and are therefore beneficial for creating nucleic acid sequences de novo.
  • the combination of an error-prone polymerase and a primer sequence serves as a writing mechanism for imparting information into a nucleic acid sequence.
  • nucleotide substrate By controlling the primer/initiator, the nucleotide substrate, or the template independent polymerase, the addition of a nucleotide to an initiator sequence or an existing nucleotide or oligonucleotide can be regulated to produce an oligonucleotide by extension.
  • these polymerases are capable of incorporating nucleotides without a template sequence and are therefore beneficial for creating nucleic acid sequences de novo.
  • the eta-polymerase (Matsuda et al. (2000) Nature 404(6781): 1011-1013) is an example of a polymerase having a high mutation rate (-10%) and high tolerance for 3' mismatch in the presence of all 4 dNTPs and probably even higher if limited to one or two dNTPs.
  • the eta-polymerase is a de novo recorder of nucleic acid information similar to terminal deoxynucleotidyl transferase (TdT) but with the advantage that the product produced by this polymerase is continuously double- stranded.
  • Double stranded DNA has less sticky secondary structure and has a more predictable secondary structure than single stranded DNA.
  • double stranded DNA serves as a good support for polymerases and/or DNA-binding-protein tethers.
  • a template dependent or template semi-dependent error prone polymerase can be used.
  • a template dependent polymerase may be used which may become error prone.
  • a template independent RNA polymerase can be used.
  • any combination of templates with universal bases can be used which encourage acceptance of many nucleotide types.
  • error tolerant cations such as Mn + can be used.
  • the present disclosure contemplates the use of error-tolerant polymerase mutants. See Berger et al., Universal Bases for Hybridization, Replication and Chain Termination, Nucleic Acids Research 2000, August 1, 28(15) pp. 2911-2914 hereby incorporated by reference. Methods of activating or inactivating template independent polymerases known to those of skill in the art are useful in the present disclosure.
  • one or more oligonucleotide sequences described herein are immobilized on a support (e.g., a solid and/or semi-solid support).
  • a support e.g., a solid and/or semi-solid support.
  • an oligonucleotide sequence can be attached to a support using one or more of the phosphoramidite linkers described herein.
  • Suitable supports include, but are not limited to, slides, beads, chips, particles, strands, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates and the like.
  • a solid support may be biological, nonbiological, organic, inorganic, or any combination thereof. Supports of the present invention can be any shape, size, or geometry as desired.
  • the support may be square, rectangular, round, flat, planar, circular, tubular, spherical, and the like.
  • the support may be physically separated into regions, for example, with trenches, grooves, wells, or chemical barriers (e.g., hydrophobic coatings, etc.).
  • Supports may be made from glass (silicon dioxide), metal, ceramic, polymer or other materials known to those of skill in the art.
  • Supports may be a solid, semi-solid, elastomer or gel.
  • a support is a microarray.
  • microarray refers in one embodiment to a type of array that comprises a solid phase support having a substantially planar surface on which there is an array of spatially defined non-overlapping regions or sites that each contain an immobilized hybridization probe.
  • substantially planar means that features or objects of interest, such as probe sites, on a surface may occupy a volume that extends above or below a surface and whose dimensions are small relative to the dimensions of the surface. For example, beads disposed on the face of a fiber optic bundle create a substantially planar surface of probe sites, or oligonucleotides disposed or synthesized on a porous planar substrate create a substantially planar surface.
  • Spatially defined sites may additionally be "addressable" in that its location and the identity of the immobilized probe at that location are known or determinable.
  • the solid supports can also include a semi-solid support such as a compressible matrix with both a solid and a liquid component, wherein the liquid occupies pores, spaces or other interstices between the solid matrix elements.
  • the semi-solid support materials include polyacrylamide, cellulose, poly dimethyl siloxane, polyamide (nylon) and cross-linked agarose, -dextran and -polyethylene glycol.
  • Solid supports and semi-solid supports can be used together or independent of each other.
  • Supports can also include immobilizing media. Such immobilizing media that are of use according to the invention are physically stable and chemically inert under the conditions required for nucleic acid molecule deposition and amplification.
  • the support structure comprises a semi-solid (i.e., gelatinous) lattice or matrix, wherein the interstices or pores between lattice or matrix elements are filled with an aqueous or other liquid medium; typical pore (or 'sieve') sizes are in the range of 100 ⁇ to 5 nm.
  • the semi-solid support is compressible.
  • the support is prepared such that it is planar, or effectively so, for the purposes of printing.
  • an effectively planar support might be cylindrical, such that the nucleic acids of the array are distributed over its outer surface in order to contact other supports, which are either planar or cylindrical, by rolling one over the other.
  • a support material of use according to the invention permits immobilizing (covalent linking) of nucleic acid features of an array to it by means known to those skilled in the art.
  • Materials that satisfy these requirements comprise both organic and inorganic substances, and include, but are not limited to, polyacrylamide, cellulose and polyamide (nylon), as well as cross- linked agarose, dextran or polyethylene glycol.
  • a polyacrylamide sheet of this type is synthesized as follows. Acrylamide and bis-acrylamide are mixed in a ratio that is designed to yield the degree of crosslinking between individual polymer strands (for example, a ratio of 38:2 is typical of sequencing gels) that results in the desired pore size when the overall percentage of the mixture used in the gel is adjusted to give the polyacrylamide sheet its required tensile properties.
  • Polyacrylamide gel casting methods are well known in the art (see Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein in its entirety by reference), and one of skill has no difficulty in making such adjustments.
  • the gel sheet is cast between two rigid surfaces, at least one of which is the glass to which it will remain attached after removal of the other.
  • the casting surface that is to be removed after polymerization is complete is coated with a lubricant that will not inhibit gel polymerization; for this purpose, silane is commonly employed.
  • a layer of silane is spread upon the surface under a fume hood and allowed to stand until nearly dry. Excess silane is then removed (wiped or, in the case of small objects, rinsed extensively) with ethanol.
  • the glass surface which will remain in association with the gel sheet is treated with ⁇ - methacryloxypropyltrimethoxysilane (Cat. No. M6514, Sigma; St. Louis, MO), often referred to as 'crosslink silane', prior to casting.
  • the only operative constraint that determines the size of a gel that is of use according to the invention is the physical ability of one of skill in the art to cast such a gel.
  • the casting of gels of up to one meter in length is, while cumbersome, a procedure well known to workers skilled in nucleic acid sequencing technology.
  • a larger gel, if produced, is also of use according to the invention. An extremely small gel is cut from a larger whole after polymerization is complete.
  • whole cells are typically cast into agarose for the purpose of delivering intact chromosomal DNA into a matrix suitable for pulsed-field gel electrophoresis or to serve as a "lawn" of host cells that will support bacteriophage growth prior to the lifting of plaques according to the method of Benton and Davis (see Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein in its entirety by reference).
  • electrophoresis-grade agarose e.g., Ultrapure; Life Technologies/Gibco- BRL
  • a physiological (isotonic) buffer e.g., fetal calf serum
  • Cells are then added to the agarose and mixed thoroughly, but rapidly (if in a bottle or tube, by capping and inversion, if in a flask, by swirling), before the mixture is decanted or pipetted into a gel tray.
  • low-melting point agarose it may be brought to a much lower temperature (down to approximately room temperature, depending upon the concentration of the agarose) prior to the addition of cells.
  • Oligonucleotides immobilized on microarrays include nucleic acids that are generated in or from an assay reaction.
  • the oligonucleotides or polynucleotides on microarrays are single stranded and are covalently attached to the solid phase support, usually by a 5'-end or a 3'-end.
  • probes are immobilized via one or more cleavable linkers.
  • the density of non-overlapping regions containing nucleic acids in a microarray is typically greater than 100 per cm 2 , and more typically, greater than 1000 per cm 2 .
  • Microarray technology relating to nucleic acid probes is reviewed in the following exemplary references: Schena, Editor, Microarrays: A Practical Approach (IRL Press, Oxford, 2000); Southern, Current Opin. Chem. Biol., 2: 404-410 (1998); Nature Genetics Supplement, 21:1-60 (1999); and Fodor et al, U.S. Pat. Nos. 5,424,186; 5,445,934; and 5,744,305.
  • Supports may be coated with attachment chemistry or polymers, such as amino-silane, NHS- esters, click chemistry, polylysine, etc., to bind a nucleic acid to the support.
  • a covalent interaction is a chemical linkage between two atoms or radicals formed by the sharing of a pair of electrons (i.e., a single bond), two pairs of electrons (i.e., a double bond) or three pairs of electrons (i.e., a triple bond).
  • Covalent interactions are also known in the art as electron pair interactions or electron pair bonds.
  • Noncovalent interactions include, but are not limited to, van der Waals interactions, hydrogen bonds, weak chemical bonds (i.e., via short-range noncovalent forces), hydrophobic interactions, ionic bonds and the like.
  • affixing or immobilizing nucleic acid molecules to the substrate is performed using a covalent linker that is selected from the group that includes oxidized 3 -methyl uridine, an acrylyl group and hexaethylene glycol.
  • a restriction site or regulatory element such as a promoter element, cap site or translational termination signal
  • Nucleic acids that have been synthesized on the surface of a support may be removed, such as by a cleavable linker or linkers known to those of skill in the art.
  • Linkers can be designed with chemically reactive segments which are optionally cleavable with agents such as enzymes, light, heat, pH buffers, and redox reagents. Such linkers can be employed to pre-fabricate an in situ solid-phase inactive reservoir of a different solution-phase primer for each discrete feature. Upon linker cleavage, the primer would be released into solution for PCR, perhaps by using the heat from the thermocycling process as the trigger.
  • affixing of nucleic acid molecules to the support is performed via hybridization of the members of the pool to nucleic acid molecules that are covalently bound to the support.
  • Immobilization of nucleic acid molecules to the support matrix according to the invention is accomplished by any of several procedures. Direct immobilizing via the use of 3'-terminal tags bearing chemical groups suitable for covalent linkage to the support, hybridization of single-stranded molecules of the pool of nucleic acid molecules to oligonucleotide primers already bound to the support, or the spreading of the nucleic acid molecules on the support accompanied by the introduction of primers, added either before or after plating, that may be covalently linked to the support, may be performed.
  • pre- immobilized primers are used, they are designed to capture a broad spectrum of sequence motifs (for example, all possible multimers of a given chain length, e.g., hexamers), nucleic acids with homology to a specific sequence or nucleic acids containing variations on a particular sequence motif.
  • sequence motifs for example, all possible multimers of a given chain length, e.g., hexamers
  • the primers encompass a synthetic molecular feature common to all members of the pool of nucleic acid molecules, such as a linker sequence.
  • the first involves the 3' capping of nucleic acid molecules with 3-methyl uridine.
  • the nucleic acid molecules of the libraries of the present invention are prepared so as to include this modified base at their 3' ends.
  • an 8% polyacrylamide gel (30:1, acrylamide: bis-acrylamide) sheet 30 ⁇ in thickness is cast and then exposed to 50% hydrazine at room temperature for 1 hour. Such a gel is also of use according to the present invention.
  • nucleic acid molecules containing 3-methyl uridine at their 3' ends are oxidized with 1 mM sodium periodate (NaI0 4 ) for 10 minutes to 1 hour at room temperature, precipitated with 8 to 10 volumes of 2% LiC10 4 in acetone and dissolved in water at a concentration of 10 pmol/ ⁇ . This concentration is adjusted so that when the nucleic acid molecules are spread upon the support in a volume that covers its surface evenly and is efficiently (i.e., completely) absorbed by it, the density of nucleic acid molecules of the array falls within the range discussed above.
  • NaI0 4 sodium periodate
  • the nucleic acid molecules are spread over the gel surface and the plates are placed in a humidified chamber for 4 hours. They are then dried for 0.5 hour at room temperature and washed in a buffer that is appropriate to their subsequent use. Alternatively, the gels are rinsed in water, re-dried and stored at -20°C until needed. It is thought that the overall yield of nucleic acid that is bound to the gel is 80% and that of these molecules, 98% are specifically linked through their oxidized 3' groups.
  • a second crosslinking moiety that is of use in attaching nucleic acid molecules covalently to a polyacrylamide sheet is a 5' acrylyl group, which is attached to the primers.
  • Oligonucleotide primers bearing such a modified base at their 5' ends may be used according to the invention.
  • such oligonucleotides are cast directly into the gel, such that the acrylyl group becomes an integral, covalently bonded part of the polymerizing matrix.
  • the 3' end of the primer remains unbound, so that it is free to interact with, and hybridize to, a nucleic acid molecule of the pool and prime its enzymatic second- strand synthesis.
  • nucleic acid molecules are crosslinked to nylon via irradiation with ultraviolet light. While the length of time for which a support is irradiated as well as the optimal distance from the ultraviolet source is calibrated with each instrument used due to variations in wavelength and transmission strength, at least one irradiation device designed specifically for crosslinking of nucleic acid molecules to hybridization membranes is commercially available (Stratalinker, Stratagene). It should be noted that in the process of crosslinking via irradiation, limited nicking of nucleic acid strands occurs.
  • nicking is generally negligible, however, under conditions such as those used in hybridization procedures. In some instances, however, the method of ultraviolet crosslinking of nucleic acid molecules will be unsuitable due to nicking. Attachment of nucleic acid molecules to the support at positions that are neither 5'- nor 3'-terminal also occurs, but it should be noted that the potential for utility of an array so crosslinked is largely uncompromised, as such crosslinking does not inhibit hybridization of oligonucleotide primers to the immobilized molecule where it is bonded to the support.
  • Supports described herein may have one or more optically addressable virtual electrodes associated therewith such that an anion toroidal vortex can be created at a reaction site on the supports described herein.
  • reagents and washes are delivered that the reactants are present at a desired location for a desired period of time to, for example, covalently attached dNTP to an initiator sequence or an existing nucleotide attached at the desired location.
  • a selected nucleotide reagent liquid is pulsed or flowed or deposited at the reaction site where reaction takes place and then may be optionally followed by delivery of a buffer or wash that does not include the nucleotide.
  • Suitable delivery systems include fluidics systems, microfluidics systems, syringe systems, ink jet systems, pipette systems and other fluid delivery systems known to those of skill in the art.
  • flow cell embodiments or flow channel embodiments or microfluidic channel embodiments are envisioned which can deliver separate reagents or a mixture of reagents or washes using pumps or electrodes or other methods known to those of skill in the art of moving fluids through channels or microfluidic channels through one or more channels to a reaction region or vessel where the surface of the substrate is positioned so that the reagents can contact the desired location where a nucleotide is to be added.
  • a microfluidic device is provided with one or more reservoirs which include one or more reagents which are then transferred via microchannels to a reaction zone where the reagents are mixed and the reaction occurs.
  • Such microfluidic devices and the methods of moving fluid reagents through such microfluidic devices are known to those of skill in the art.
  • Immobilized nucleic acid molecules may, if desired, be produced using a device (e.g., any commercially-available inkjet printer, which may be used in substantially unmodified form) which sprays a focused burst of reagent-containing solution onto a support (see Castellino (1997) Genome Res. 7:943-976, incorporated herein in its entirety by reference).
  • a device e.g., any commercially-available inkjet printer, which may be used in substantially unmodified form
  • Such a method is currently in practice at Incyte Pharmaceuticals and Rosetta Biosystems, Inc., the latter of which employs "minimally modified Epson inkjet cartridges" (Epson America, Inc.; Torrance, CA).
  • the method of inkjet deposition depends upon the piezoelectric effect, whereby a narrow tube containing a liquid of interest (in this case, oligonucleotide synthesis reagents) is encircled by an adapter.
  • An electric charge sent across the adapter causes the adapter to expand at a different rate than the tube, and forces a small drop of liquid reagents from the tube onto a coated slide or other support.
  • Reagents can be deposited onto a discrete region of the support, such that each region forms a feature of the array.
  • the feature is capable of generating an anion toroidal vortex as described herein.
  • the desired nucleic acid sequence can be synthesized drop-by-drop at each position, as is true for other methods known in the art. If the angle of dispersion of reagents is narrow, it is possible to create an array comprising many features. Alternatively, if the spraying device is more broadly focused, such that it disperses nucleic acid synthesis reagents in a wider angle, as much as an entire support is covered each time, and an array is produced in which each member has the same sequence (i.e., the array has only a single feature).
  • Exemplary embodiments of the present disclosure are directed to methods of enzymatic synthesis of user-defined nucleic acid sequences using TdT.
  • the methods according to the present disclosure contemplate four major parts: physical and chemical control of nucleic acid polymer exposure to NPU, nucleotide analogue substrates for TdT, conditions for TdT polymerization, and finally, an example implementation of our inventions for TdT-based NPUs to synthesize nucleic acids of a defined information content.
  • These novel methods according to the present disclosure can be used for the synthesis of nucleic acid polymers for information storage in DNA.
  • novel methods according to the present disclosure further provide improved control of the number and nature of nucleotides that template-independent DNA polymerases, such as TdT, incorporate into nucleic acid polymers and enable user-defined synthesis of nucleic acid sequences useful for biological applications.
  • template-independent DNA polymerases such as TdT
  • the present disclosure provides that limiting the number of nucleotide additions by TdT can be achieved by controlling one or a combination of the following three elements of the polymerization reaction: the primer/initiator, the nucleotide substrate, or the polymerase.
  • Previous and ongoing attempts at custom DNA synthesis using TdT focus on the primer/initiator combined with the nucleotide. Specifically, others have tried using reversible- terminator nucleotide analogs to synthesize DNA of a desired sequence. However, it has been found that TdT does not efficiently work with any of the available reversible terminator nucleotide analogues. Furthermore, using such analogues adds to both the cost and complexity of DNA synthesis while increasing synthesis time.
  • the present disclosure provides novel methods of enzymatic synthesis that focus on controlling the nucleotide and the polymerase, i.e., the NPU, using various physical and chemical control approaches.
  • the time the initiator/primer is in physical contact with NPU is controlled.
  • the method of the disclosure provides an efficient way to establish such a physical method of NPU exposure control through fluidics.
  • An exemplary embodiment of such a method is shown in FIGS. 1A and IB, in which initiator oligonucleotides were immobilized on a surface of a fluidic device (called initiator patch or patch).
  • the patch is then exposed to NPUs which include TdT pre-mixed with only one of the four possible nucleotide triphosphates (dNTPs).
  • dNTPs nucleotide triphosphates
  • the microfluidic device flows each NPU over the patch at a given rate, thus limiting the exposure time of the patch to each NPU to generate an extension of a desired base to a desired count or desired distribution of counts.
  • the device also allows control over the order of NPUs, thereby allowing control over the incorporated sequence and information content (FIG. 1A). For instance, for the synthesis of the sequence "GATC,” the patch will be serially exposed to four NPUs. The NPUs will each includes TdT with dGTP, dATP, dTTP, and dCTP, respectively.
  • the fluidic control exposes the patch to each of the NPUs for the optimal time which ensures addition of that specific nucleotide to the entire patch of initiator oligonucleotides.
  • the amount of time the nucleotide substrates are available to the enzyme for extension of the primer/initiator is controlled.
  • the enzyme "ATP diphosphohydrolase" (Apyrase), which degrades dNTPs, was added to a reaction with TdT (FIG. 2A). This addition results in two competing reactions: one is the polymerization of free nucleotides by TdT to a nucleic acid polymer and the other is the degradation of free nucleotides available to TdT by Apyrase.
  • the concentration of Apyrase was optimized which allowed reproducible addition of nucleotide extensions of set lengths.
  • nucleotide Once a nucleotide was added to the initiator by the polymerase and its excess was degraded by apyrase, the next nucleotide would be added to the mixture (FIG. 2B).
  • the initiator, TdT, and apyrase were mixed. Then a small amount of dGTP was added to this mix. After a few seconds, once TdT has extended the initiators and apyrase has degraded the excess dGTP, a small amount of dATP would be added to the mix. So on, a few seconds later dTTP would be added. dCTP would be added a few seconds after that.
  • This new control strategy obviated the challenging requirement for high temporal exposure control presented in the first physical control approach.
  • these additional approaches include: activating inactive nucleotides or inactive TdT enzyme by heat, wavelengths of light, or pH, and using a protease to remove the TdT enzyme as opposed to removing the dNTPs by apyrase from the reaction after a set amount of time, etc.
  • extension efficiency, rate, and extension length distribution of each of the four natural nucleotides was different.
  • dCTP shows the most optimal behavior while dATP and dTTP show the poorest behavior with respect to TdT-based DNA synthesis.
  • nucleotide analogues were screened to search for nucleotide analogues with a superior performance compared to their natural counterparts in TdT-based DNA synthesis. The following nucleotide analogues with the NPU formulation which included Apyrase were explored.
  • nucleotide analogues displayed equally good or superior efficiency, rate, and/or length distribution compared to their natural counterparts with TdT (see FIG. 3 for nucleotide structures).
  • 5-Propargylamino-dUTP In general, it has been observed that all nucleotide analogues that are more positively charged than their natural counterparts are far more efficient substrates of TdT. These analogues include, but are not limited to, 5-Aminoallyl-dUTP and 5-Propargylamino-dUTP.
  • nucleotide analogues within the scope of the present disclosure include 1 - 5-Hydroxy-dCTP (hdCTP)
  • the present disclosure provides exemplary apparatus, protocol, and implementation of a method of NPU enzymatic synthesis to generate a nucleic acid polymer of a given information content.
  • An embodiment of this implementation is illustrated in FIG. 4.
  • Each of the four NPUs is a formulation of TdT, Apyrase, and one of the following nucleotides: 7- Deaza-7-bromo-dATP, dCTP, dGTP, and 7-propynyl-dUTP.
  • a robotic dispensing system (Mantis Robot from the company Formulatrix) which can be programmed to reproducibly move in xyz space and to dispense liquids at 100 nanoliter increments was used.
  • 100 nanoliters of initiator oligos were deposited on Arraylt SuperAldehyde2 coated slides (Cat. SMA2F) at an optimal concentration between 0.04 micromolar and 5 micromolar, and resuspended in IX Microspotting solution (Arraylt).
  • the initiator oligos were designed with a 5 prime amine modification for immobilization to the glass surface and with deoxyUridines to enable oligo release by USER (Uracil-Specific Excision Reagent) Enzyme as seen:
  • the slides with the initiator oligos were incubated with 1.4X Microspotting solution (Arraylt) and 500 millimolar NaCl (3.5mililiter 2x oligo spotting solution, 1 milliliter water, 0.5 milliliter of a 5 molar NaCl solution) for 24 hours in a closed environment to prevent evaporation.
  • the slides were then dried first at room temperature then incubated at 60°C for 1 hour.
  • the slide were then washed once with water, once with 0.1% SDS+1 millimolar Tris HC1 at pH 8.0, and three more times with water (with vigorous shaking the last time).
  • the slides were incubated in 80°C water for three minutes and then submerged in ice-cold 100% ethanol for thirty seconds (last sequence to denature the oligos and keep them that way).
  • the slides were ready to be used for enzymatic synthesis once dried by centrifugation at 500g for 3 minutes in a conical tube.
  • a DNA adapter was ligated to the 3 prime end of the extended oligos. Ligation was carried out on the slides overnight at room temperature by flooding the slide surface with the following mixture in a sealed container to prevent moisture and oxygen:
  • This ligation mixture was washed off the slides by 0.1% SDS wash and two washes with water and subsequently dried by ligation.
  • Samples can be eluted by depositing 1 microliter of the following USER mix:
  • an initiator oligonucleotide was first immobilized onto a surface called the initiator patch, in this exemplary embodiment by UV crosslinking DNA to glass surface. After tethering the initiator to a solid support, the surface was treated to neutralize the chemical reactive groups such as aldehydes in this case, and to increase hydrophobicity. A pre-fabricated custom PDMS device was subsequently plasma treated in order to covalently attach it to the glass slide with the initiator patch.
  • Nitrogen pumps were used to push aqueous slugs, i.e. discrete volumes, containing the enzymatic cocktail, through one channel and oil slugs, i.e. discrete volumes, containing the wash solution, through the other channel.
  • the pump rates were adjusted to give roughly
  • each aqueous slug on the initiator patch was adjusted by altering the pump rates, while keeping the relative rates of the aqueous channel and oil pumps the same.
  • the total reaction time was the sum of all residence times of each aqueous slug passing over the initiator patch while the pumps are active.
  • the synthesized DNA on the initiator patch can be assessed for the number of nucleotides added. By imaging, the length of nucleotides added can be assessed by the use of fluorescent nucleotides or hybridization of fluorescent probes.
  • the synthesized DNA on the initiator patch can also be released, in this exemplary embodiment, enzymatically, by the use of USER (Uracil-Specific Excision Reagent) enzyme which cleaves the uracils that are near the 5 prime distal end of the initial initiator oligonucleotide.
  • USER User-Specific Excision Reagent
  • the cleaved DNA strands are then collected by flow into a tube.
  • These strands are subsequently PCR amplified and the number of synthesized nucleotides evaluated by two methods: electrophoresis on agarose or PAGE gels for cursory bulk analysis and sequenced with next-generation sequencing platforms such as Illumina, Pacific Bioscience, and/or Oxford Nanopore for quantitative single-molecule analysis.
  • Oligo ctgac was diluted to 5uM in PBS. 3uL of this oligo solution was placed on the glass slides and incubated at 60°C until dry.
  • oligo was covalently attached to the glass by exposure to lOOuJ for 6.5 seconds. The slides were then washed with 0.1XSSC with 0.2% SDS for 5 minutes with shaking, rinsed once with water and dried at 60°C for lOmin. 4. A PDMS mask with, previously casted to contain a 100 micron channel with a standard T junction (illustration in FIG. 5) was treated with Oxygen Plasma at 200 milliwatts for 30 seconds and sealed onto the glass slides such that the oligo patch would be under the part of the channel that was intended for the initiator.
  • the organic phase was a 7 to 3 mixture of Hexadecane and AR-20 Silicon Oil (both obtained from Sigma). An air pump was used to continuously pump this organic/wash solution in the device through the Organic /Wash Input with a back pressure of 10 Mega Pascals.
  • the aqueous reagent was prepared with the following composition:
  • Another airpump was used to continuously pump this aqueous reagent solution through the Aqueous/Reagent Input on the device.
  • the amount of polymerization on the initiator patch was assessed by imaging the initiator patch of the device in Cy5 channel using an inverted fluorescence microscope.
  • an initiator oligonucleotide was first immobilized onto a surface, for example with the use of a 5 prime modified oligonucleotide onto aldehyde functionalized glass slides. After tethering the initiator to a solid support, the surface was treated to neutralize the chemical reactive groups such as aldehydes in this case, and to increase hydrophobicity.
  • Each of these nucleotides were printed on an oligo spot with a liquid handling robot followed by printing of the enzymatic mix comprising TdT (Terminal deoxynucleotidyl transferase) and Apyrase. After a specified reaction time between the oligonucleotide, printed nucleotide, and print enzymatic mix, the slides were washed and a terminal oligonucleotide was ligated onto the synthesized DNA to allow for subsequent amplification.
  • TdT Terminal deoxynucleotidyl transferase
  • Each of the synthesized DNA strands were released off the solid-support surface, in this exemplary embodiment, enzymatically, by the use of USER (Uracil-Specific Excision Reagent) enzyme which cleaves the uracils that are near the 5 prime distal end of the initial initiator oligonucleotide.
  • USER User-Specific Excision Reagent
  • These strands were subsequently PCR amplified and the number of synthesized nucleotides were evaluated by two methods: electrophoresis on agarose or PAGE gels for cursory bulk analysis and sequenced with next-generation sequencing platforms such as Illumina, Pacific Bioscience, and/or Oxford Nanopore for quantitative single-molecule analysis.
  • a 2 ⁇ droplet of each nucleotide concentration was printed on an oligo spot with a liquid handling robot and then dried at room temperature (RT) for a few minutes.
  • oligo 5P-cagtc has the following sequence:
  • Ligation mix was washed by 0.1 %SDS wash and two washes with water. The slide was dried by centrifugation.
  • the number of nucleotides added to the oligonucleotide initiator were quantified for each nucleotide type and concentration in addition to the number of oligonucleotide initiators that received nonzero addition of nucleotides for each nucleotide type and concentration (See, FIG. 7 and FIG. 8), where 0 to 7 for each base (i.e. AO to A7) corresponds to increasing concentrations of a given nucleotide.
  • the objective of these experiments is to determine the range of pH in which TdT is active.
  • a buffer of 50mM Tris (base) and 50mM Boric acid was prepared and its pH (initially at -8.5) was adjusted to 6.05, 6.9, 7.93, 8.96, 10.02, and 11.07 using acetic acid and sodium hydroxide. These buffers serve as 2X buffers in the experiments.
  • the results in the TBE-Urea gel established that pH can be used to regulate the activity of TdT enzyme in a way that is adaptable for pH-based TdT-control for data storage. It also needs to be established that the effects of pH on TdT are reversible; that is, the enzyme's activity can be substantially reduced at an unfavorable pH but can be reverted back to normal activity at favorable pH. The ensuing experiment was performed to evaluate this question.
  • TdT was incubated at a pH for 15 minutes without the nucleotide or the initiator, it was then combined with the nucleotide and the initiator in such a way that the final pH of the mixture during polymerization would be different from that of the initial 15min incubation.
  • a buffer of 50mM Tris (base) and 50mM Boric acid was prepared and its pH (initially at -8.5) was adjusted to 6.05, 6.9, 7.93, 8.96, 10.02, and 11.07 using acetic acid and sodium hydroxide. These buffers serve as 2X buffers in the experiment.
  • TdT is highly active in pH ranges that are above 6 and below 11, and it is substantially inactivated at pH ranges that are below 6 and above 11. It was clear from this experiment that the enzymatic activity of TdT could be reversibly inhibited by both increasing and reducing the pH. Inhibition of TdT enzymatic activity was more effective at pH ⁇ l 1 than it was at pH ⁇ 6.
  • the starting pH was 6 and 11 respectively where the enzyme showed little to no polymerization activity.
  • the following modified nucleotide analogues were tested with an initiator oligonucleotide: 5-Hydroxy-dCTP (hdCTP), 5-Hydroxymethyl-dCTP (hmdCTP), 5-Bromo- dCTP (BrdCTP), 5-Iodo-dCTP (IdCTP) and 5-methyl-dCTP (5m-dCTP).
  • hdCTP 0, 2, 4, 8, 16, 32uM
  • hmdCTP 0, 2, 4, 8, 16, 32uM
  • BrdCTP 0, 2, 4, 8, 16, 32uM
  • IdCTP 0, 2, 4, 8, 16, 32uM
  • 5mdCTP 0, 2, 4, 8, 16, 32uM.
  • the reaction stop solution, STOP&LOAD was prepared as 20ul Novex 2X TBE-Urea loading buffer containing 20mM EDTA.
  • a 16ul reaction solution was prepared as follows as a mastermix: 3 ⁇ 1 Water; ⁇ 2X Reaction Buffer; 2 ⁇ 1 luM Primer; ⁇ TdT enzymatic mix; for a total volume of 16ul.
  • dNTP variable 4X
  • Fig. 15 is a gel image showing results for 5BR-dCTP and 5I-dCTP.
  • Fig. 16 is a gel image showing results for 5h-dCTP and 5hm- dCTP.
  • Fig. 17 is a gel image showing results for 5m-dCTP and dCTP.

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Abstract

L'invention concerne des procédés de fabrication d'un polynucléotide dans lequel l'addition de nucléotides peut être commandée physiquement, chimiquement et/ou enzymatiquement. Les procédés consistent à combiner un certain nucléotide, des cations, une ADN polymérase sensible aux erreurs ou indépendante de la matrice au niveau d'un site de réaction comprenant une séquence d'initiation fixée à celui-ci et ayant un nucléotide de terminaison 3', les réactifs de réaction pouvant être modulés et dans des conditions qui permettent une addition covalente d'un ou de plusieurs d'un certain nucléotide au nucléotide à terminaison 3' de telle sorte que ledit certain nucléotide devienne un nucléotide à terminaison 3', et répéter l'étape d'addition jusqu'à ce que le polynucléotide soit formé.
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